Navigation Links
miRNA Expression in White Blood Cells

Emmanuel Labourier, Marianna Goldrick, Ambion, Inc.


microRNAs (miRNAs) are endogenous, 21-24 nt RNAs that mediate post-transcriptional gene regulation by pairing with the 3' untranslated region of messenger RNAs and acting as translational repressors. The study of miRNA is an emerging and exciting area of research with applications for basic, applied, and therapeutic science. Recent reports suggest a role for miRNAs in development, cell differentiation, apoptosis, and cancer. Ambion has recently developed several new tools for isolation and detection of miRNAs, which were used to investigate miRNA expression in white blood cells.


Introduction
In the past few years, several discoveries have indicated that miRNAs play a role in various cellular functions. Early studies of miRNAs suggest that they could become a valuable tool for elucidating how gene expression can direct development, differentiation, and proliferation. This area of research could provide more insight into the causes of certain diseases, leading to the identification of novel therapeutic or diagnostic targets. Interest in the identification, detection, and use of small RNA molecules has exploded in response to these discoveries. However, standard RNA isolation and quantitation techniques have been optimized for larger RNAs and are not always favorable for recovering and analyzing smaller RNA species.


Analysis of Small RNA Expression
The size, and in some cases the low expression levels, of small RNAs can make their analysis difficult. The small size of these molecules alone precludes the use of RT-PCR as a detection method, since they are too short to be specifically primed for PCR-mediated amplification. Furthermore, most RNA isolation procedures have been optimized to recover long (>500 nt) messenger RNAs, while ignoring smaller molecules. As a result, conventional RNA extraction methods can result in the loss of substantial amounts of small RNAs from samples. To address the issues of small RNA recovery and detection, Ambion scientists have developed the mirVana line of products, a group of kits designed to provide a complete solution for small RNA analysis.

The mirVana miRNA Isolation Kit (patent pending) was codeveloped with the mirVana miRNA Detection Kit to provide quantitative yields of small RNAs (including miRNA, siRNA, tRNA, and rRNAs) from virtually any biological sample. The patented mirVana miRNA Detection Kit is a set of reagents for small-volume solution hybridization assays that is far more efficient than membrane-based hybridization (e.g. Northern blots). The assay can detect relatively abundant miRNA species, such as miR-124 in brain, in as little as 10 ng of total RNA.

An example of small RNA analysis using these tools is shown in Figure 1. The data show that the same amount of 5.8S and 5S rRNA was recovered from mouse brain, kidney, liver and thymus. In contrast, significant variations in miRNA levels were observed in these tissues. For example, let-7 was more abundant in brain and kidney, and miR-16 was highly expressed in thymus only. As expected, based on published reports [1, 2], miR-124 was detected only in the brain sample.

Figure 1. Analysis of miRNA Expression Across Mouse Tissues. Total RNA was isolated from ~100 mg of the indicated tissues with the mirVana miRNA Isolation Kit. Purified RNA (1 g) was analyzed on a 15% denaturing polyacrylamide gel stained with ethidium bromide (left panel). let-7 miRNA, and 5.8S and 5S rRNA were detected by Northern blot. miR-16 and miR-124 miRNA were detected by solution hybridization with the mirVana miRNA Detection Kit. All probes were labeled and purified with the mirVana Probe & Marker Kit.


Expression of miRNA in Blood Cells
Although miRNAs have been found in all mammalian tissues examined so far, there is little if any published information about miRNA expression in blood. This may be due to the inherent difficulty in isolating high quality RNA from human blood. To examine the efficiency of the mirVana system for use in analysis of miRNAs in white blood cells, blood was collected from 14 healthy volunteers. Leukocytes were fractionated from whole blood and the total RNA population, including miRNA, was extracted using the mirVana miRNA Isolation Kit. The RNA recovered from the fractionated white blood cells was quantitated by UV absorbance and analyzed by ethidium bromide staining on a 15% denaturing polyacrylamide gel. RNA yields ranged from about 20-40 g per 10 ml blood sample. No obvious degradation of the ribosomal RNA and tRNA bands was observed. Four representative samples are presented in Figure 2.

Figure 2. Analysis of miRNA Expression in White Blood Cells. Leukocytes were collected from 10 ml EDTA-anticoagulated blood samples from four different donors, and total RNA was isolated with the mirVana miRNA Isolation Kit. Samples were analyzed as described in Figure 1 except that a probe specific for miR-22 miRNA was used in the solution hybridization assay.


The RNA samples were then analyzed for the presence of several small RNAs using Northern blot or the mirVana miRNA Detection Kit. Interestingly, several miRNAs--let-7, miR-16, and miR-22--were detected in total RNA from white blood cells (Figure 2). miR-16 was the most abundant miRNA in all of the 14 samples. A direct comparison of total RNA samples from other human tissues showed that miR-16 is expressed at a higher level in white blood cells than in any other tissues we have examined (data not shown). While different expression levels were observed across donors for the few miRNA tested, there was no clear donor-specific difference in the global miRNA expression pattern. Experiments are in progress to determine whether other miRNAs may have donor-specific expression patterns and whether these patterns vary over time.


Outlook
As the study of small RNA molecules continues, the need for fast, robust, and sensitive miRNA analysis tools is increasing. Further investigation of the biological role of miRNA will be facilitated by methods such as those incorporated in the mirVana system. High throughput detection of miRNA will undoubtedly help to unravel their complex spatial and temporal expression patterns, as well as determine variations in miRNA levels during tissue development and in disease states.


back to top


Ordering Information
Cat# Product Name Size 1550 mirVana miRNA Probe Construction Kit 30 rxns 1552 mirVana miRNA Detection Kit 100 rxns 1554 mirVana Probe & Marker Kit 30 rxns + 10 marker rxns 1560 mirVana miRNA Isolation Kit up to 40 purifications
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Cancer-Related miRNAs Uncovered by the mirVana miRNA Microarray Platform
2. Detecting miRNA & siRNA
3. Isolate It All: siRNA miRNA Total RNA Native Protein
4. Get the miRNA OutPlant Applications
5. Novel MicroRNA Array Technology for Sensitive miRNA Profiling
6. pSilencer 4.1-CMV: Versatile Vectors for Expression of siRNA, miRNA, and mRNA
7. Precursor miRNAs for Successful miRNA Functional Studies
8. Prepare siRNA and miRNA Probes in Just 1 Hour
9. Reagents for Up and Down-Regulation of miRNA Activity in Mammalian Cells
10. An Epitope Tagging Vector for Gene Expression in Mammalian Cells
11. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: miRNA Expression White Blood Cells

(Date:10/22/2014)... (PRWEB) October 21, 2014 The North ... concerned market North America with analysis and forecast of ... 2013, and is expected to reach $623.6 million by ... 2018. , Browse through the TOC of the North ... of the in-depth analysis provided. This also provides a ...
(Date:10/22/2014)... 2014 Grace Century, a private equity ... announces the addition of Dr. Yousef “Josh” Siddiqui ... team. Dr. Siddiqui will provide further healthcare expertise to ... of University College Medical School in the ... in 2001. With further certification as a General Practitioner ...
(Date:10/22/2014)... 2014 Nuvilex, Inc. (OTCQB: NVLX) – According ... worldwide are living with diabetes, with  that number expected ... global market for diabetes treatments is approximately $500 billion.  ... died from pancreatic cancer.  Pancreatic cancer is the fourth ... the United States , and according ...
(Date:10/20/2014)... 2014 Convey Computer , the ... from Iowa State University won first place in the ... Convey HC-2ex, the team’s solution achieved the highest overall ... the second place finisher. , Experts from all segments ... month long challenge, using a variety of design tools, ...
Breaking Biology Technology:The North American crystal oscillator market is expected to reach $623.6 million by 2018 - New Report by MicroMarket Monitor 2The North American crystal oscillator market is expected to reach $623.6 million by 2018 - New Report by MicroMarket Monitor 3The North American crystal oscillator market is expected to reach $623.6 million by 2018 - New Report by MicroMarket Monitor 4Dr. Yousef Siddiqui joins the Grace Century Advisory Team 2Nuvilex Brief Analyst Report: Thinking Outside the Box by BrokerBank Securities, Inc. 2Team from Iowa State Wins 2014 MemoCODE Design Contest Using Convey HC-2ex 2Team from Iowa State Wins 2014 MemoCODE Design Contest Using Convey HC-2ex 3
... Yves Lionel Assant is,the new Chairman of Gerresheimer,s ... was already a member of the Supervisory Board. ... position for personal reasons. He,remains a member of ... & life science supply and services,company with market ...
... Stephen Simes, chief executive,officer of BioSante Pharmaceuticals, ... be featured in an exclusive interview with, http://www.wallst.net ... EDT. The interview,will be posted on http://www.wallst.net ... The interview will cover topics including BioSante,s ...
... Calif., Sept. 24 Today, the rights ... utilize PARI,s,eFlow electronic nebulizer for aerosol delivery ... of Corus Pharma in,August 2006, this is ... eFlow is featured prominently in the development ...
Cached Biology Technology:WallSt.net Announces Upcoming Interview with CEO of BioSante Pharmaceuticals 2Pediatric Asthma Programs Featuring PARI Pharma's eFlow Acquired by AstraZeneca 2
(Date:10/14/2014)... Mount Sinai-led research team has discovered a new kind of ... a cell that lines liver blood vessels, according to a ... . The existence of such a cell type contradicts current ... embryo, and may hold clues to origins of, and future ... from a single cell into a complex being made up ...
(Date:10/14/2014)... CA – October 14, 2014 – Scientists from The ... from the National Institutes of Health (NIH) to lead ... virus-induced hemorrhagic fever disease in Africa. The study aims ... why some patients die, while others survive the inflection. ... understand the basic mechanism of how Lassa fever virus ...
(Date:10/14/2014)... international team of researchers, led by the Chinese Academy ... Nature Genetics a brief genomic history of ... the tomato plant. , The C.M. Rick Tomato Genetics ... in this study by providing seed of both cultivated ... which builds on the first tomato genome sequence completed ...
Breaking Biology News(10 mins):Stem cell discovery challenges dogma on how fetus develops; holds insights for liver cancer and reg 2Stem cell discovery challenges dogma on how fetus develops; holds insights for liver cancer and reg 3Scripps Research Institute team receives $6.6 million to investigate deadly Lassa virus 2
... Department of Energy,s Savannah River National Laboratory (SRNL) ... University to decrease mold growth in flooded homes ... by the Department of Homeland Security (DHS) through ... University asked SRNL and Tuskegee University to join ...
... JOLLA, Calif., July 14, 2010 Put simply, a tumor ... the cell cycle the cell,s process of duplicating itself ... of proteins tells other proteins what to do and when ... regulation fail, cell growth can get out of hand. A ...
... -- The 52nd meeting of the American Association ... - 22, 2010 in Philadelphia, PA. AAPM ... broadly-based scientific and professional discipline encompassing physics principles ... includes medical physicists who specialize in research that ...
Cached Biology News:SRNL works to decrease hazards from mold in water damaged homes 2New role for the JNK protein 2Nanotech medicine, tumor tracking, new technologies and more 2Nanotech medicine, tumor tracking, new technologies and more 3Nanotech medicine, tumor tracking, new technologies and more 4Nanotech medicine, tumor tracking, new technologies and more 5Nanotech medicine, tumor tracking, new technologies and more 6Nanotech medicine, tumor tracking, new technologies and more 7Nanotech medicine, tumor tracking, new technologies and more 8Nanotech medicine, tumor tracking, new technologies and more 9Nanotech medicine, tumor tracking, new technologies and more 10Nanotech medicine, tumor tracking, new technologies and more 11Nanotech medicine, tumor tracking, new technologies and more 12Nanotech medicine, tumor tracking, new technologies and more 13Nanotech medicine, tumor tracking, new technologies and more 14Nanotech medicine, tumor tracking, new technologies and more 15Nanotech medicine, tumor tracking, new technologies and more 16Nanotech medicine, tumor tracking, new technologies and more 17